1. Field of the Invention
This invention relates to improved ultra-sensitive metallic silver stains for polypeptides, especially when fixed in synthetic gels, particularly polyacrylamide gels.
2. Description of the Prior Art
Detection and characterization of polypeptides is of fundamental importance to many areas of biology and clinical medicine. In some endeavors, such as genetic screening for mutational events, monitoring for pathophysiologic changes in disease states, and the diagnosis of genetic diseases, the efficiency of the search is directly proportional to the number of polypeptides that can be detected and characterized in cellular extracts of body fluids. Additionally polypeptides, hormones, etc., that are present in trace amounts are often of great importance for various medical reasons.
Electrophoresis (defined generally as the movement of charged particles in solution under the influence of an electrical field), is a primary laboratory detection and characterization technique, especially useful for polypeptides and other micromolecules, such as nucleic acids. It is also useful in separating small particles such as viruses, cells, sub-cellular organelles and organic molecules such as steroids and amino acids.
Continuing developments in two-dimensional gel electrophoresis have provided the capability of resolving thousands of polypeptides from complex biological mixtures. However, the inability to detect polypeptides present in low concentration has limited the application of this technology, particularly in clinical screening for pathological states, endocrinology, mammalian metabolism, developmental biology, and immunology.
Because the improved gel electrophoretic techniques greatly increase polypeptide resolution, visual detection methods employing conventional polypeptide dyes are no longer adequate.
The most commonly used conventional polypeptide stain is Coomassie Blue, which may be considered as a prototype. Dyes of this type are mainly dependent upon the electrostatic attraction between dye and polypeptide, stabilized by van der Wall's forces. In fact, Coomassie Blue and a variety of other dyes exhibit particular affinities for polypeptides of specific charge. Coomassie Blue, an acidic dye, stains basic polypeptides most intensely, while crystal violet is the most effective stain for acidic polypeptides. Other dyes for which quantitative aspects of staining have been investigated include Amido Black, Fast Green, and Fe.sup.2+ -bathophenanthroline sulfonate. In contrast, the Remazol Brilliant Blue R method depends on a covalent bond between dye and polypeptide. With Coomassie Blue, linearity has been found, by staining for 30 minutes in 1.5 mm diameter gels, in the polypeptide concentration range of 0.05-2 .mu.g using the parameter of relative spot area. Staining for 60 rather than 30 minutes may result in an increase in the slope of the area/concentration relationship and nonlinearity due to saturation above 1 .mu.g. Fluorescamine can react with terminal and .epsilon.-amino groups of polypeptides in gels to achieve a sensitivity at least equal to that of Coomassie Blue, with linearity from 1 to 7 .mu.g "per spot". MDPF (2-methoxy-2-4-diphenyl-3(2H)-furanone) may be used to label polypeptides fluorescently prior to electrophoresis, with linearity from 10 ng to 10 .mu.g of protein. However fluorescent staining of polypeptides prior to electrophoresis may alter their electrophoretic patterns.
An assortment of other techniques which do not require modification of polypeptides prior to electrophoresis also exist. These include densitometric scanning for absorbance at 280 nm, binding of radiolabelled or fluorescent ligands such as concanavalin A to glycoproteins, binding of antisera to polypeptides at the gel surface, and staining of specific polypeptide moieties including carbohydrate sidechains with PAS, sulfhydryl groups with 5,5'-dithiobis (2-nitrobenzoic acid), copper polypeptides with cyanide-tetrazolium, cadmium polypeptides with dipyridyl-ferrous iodide and Ca.sup.2+ -polypeptides with .sup.45 Ca autoradiography.
Radioactive detection techniques offer a higher degree of sensitivity than the stains but are often impractical to use. In vivo radiolabelling may alter cellular metabolism and it may be impossible to label certain human polypeptides. In vitro radiolabelling has the disadvantage that it might alter the electrophoretic mobility of polypeptides. Furthermore, radioactive reagents sometimes prove too expensive and long exposure to detect trace polypeptide may result in the problem of "autoradiographic spreading".
Polypeptides labelled with a radioactive precursor may be detected by autoradiography and/or fluorography, which have been standardized and used quantitatively. A set of radiographic standards placed next to the gel during exposure of the film may facilitate quantification. Fluorography requires impregnation of the gel with a scintillation fluor and is of greatest use when a low energy beta emitter has been used for labelling or when an increase in sensitivity of detection is required. Quantitative use of fluorography requires prefogging of the film. Recently, in vitro methods for chemically radiolabelling polypeptides prior to electrophoresis have been described. These include the reductive methylation of .epsilon.-amino groups of lysyl residues and .alpha.-amino groups of N-terminal amino acids which can be accomplished with formaldehyde and sodium cyanoborohydride. Carbon.sup.14 formaldehyde or .sup.3 H sodium cyanoborohydride may be used as the radiolabel. N-succinimidyl [2,3-.sup.3 H]propionate can be used to label covalently terminal and .epsilon.-amino groups either in vivo or in vitro. All of these methods may, unfortunately, alter the labelled polypeptides' mobility.
The above staining methods, moreover, are difficult to perform, hazardous, time consuming, and unless the polypeptides are heavily labeled, lack the sensitivity to detect proteins present in low or trace concentrations. A problem arises, for example, with body fluids, such as cerebrospinal and amniotic fluids, which are often difficult to obtain in quantity and frequently contain certain abundant proteins which cause distortion of electrophoretic patterns when sufficient sample is analyzed to observe specific trace polypeptides.
Recently, highly sensitive silver stain methods for polypeptides in polyacrylamide gels have been developed. These methods have the disadvantages of being too wasteful of silver and/or being too complicated, and in most instances are less sensitive or reproducible than the improved method of this invention, although more sensitive than non-silver stains.
Kerenyl and Gallyas, in "A Highly Sensitive Method for Demonstrating Proteins in Electrophoretic, Immunoelectrophoretic and Immuno-diffusion Preparations", Clin. Chim. Acta, 38, 465-467 (1972) discloses a silver stain for proteins in agar gel in which the gel is immersed in potassium ferrocyanide, and then in a two solution developer containing sodium carbonate and water in the first solution and ammonium nitrate, silver nitrate, tungsto-silicic acid, and formaldehyde in the second solution. The possibility of using polyacrylamide gel is mentioned.
Kerenyi and Gallyas, in "Uber Probleme der Quantitiven Auswertung der mit Physikalischer Entwicklung Versilberten Agarelektrophoretogramme", Clin. Chim. Acta, 47, 425-436 (1973) continued the study of the silver stain disclosed in 1972, above. Artifacts developed during the staining, whose avoidance is discussed.
Veerheecke, in "Agargel Electrophoresis of Unconcentrated Cerebrospinal Fluid", J. Neurol., 209, 59-63 (1975) discloses silver staining in agar gel utilizing two solutions after immersion of the protein-containing gel in potassium ferrocyanide. The first solution contains sodium carbonate in water, the second solution contains ammonium nitrate, water, formaldehyde, and tungsto-silicic acid as well as silver nitrate. The results reported are mixed, although generally favorable. Mention is made that the method of Kerenyi and Gallyas (1972), supra, of which this was a replication, did not appear to have found widespread acceptance, possibly because discrete bands in the gamma region of the electropherogram could not be detected and because numerous artifacts were experienced. Veerheecke himself experienced difficulties with bands in several regions.
Karcher, Lowenthal and Van Soom, in "Cerebrospinal Fluid Proteins Electrophoresis without Prior Concentration", Acta nuerol. Belg., 79, 335-337 (1979), discloses silver staining utilizing two solutions after immersion of the protein-containing gel in potassium ferrocyanide. The first solution contains sodium carbonate, the second solution contains ammonium nitrate, water, formaldehyde, and tungsto-silicic acid as well as silver nitrate. The disclosure concludes that the stain is comparable to that obtained for conventional electrophoresis staining with amido-black, working with concentrated cerebrospinal fluid.
Switzer, Merril and Shifrin, in "A Highly Sensitive Silver Stain for Detecting Proteins and Peptides in Polyacrylamide Gels", Anal. Biochem, 98, 231-237 (1979), discloses a silver stain in which the proteins are fixed by soaking of the gel in various methanol/acetic acid mixtures for at least 2.5 hours, soaking the gel in a paraformaldehyde solution for 0.5 hours, placing the gel in a cupric nitrate/silver nitrate solution for at least 0.5 hours, placing the gel in a diammine solution (a mixture of silver nitrate, NaOH, NH.sub.4 OH, and ethanol) for 10 min., and twice reducing the gel stain with formaldehyde and citric acid. The stain was found to be 100 times more sensitive than Coomassie Blue and comparable to autoradiography.
Merril, Switzer, and Van Keuren, in "Trace Polypeptides in Cellular Extracts and Human Body Fluids Detected by Two-Dimensional Electrophoresis and a Highly Sensitive Silver Stain", Proc. Natl. Acad. Sci. U.S.A., 76, 4335-4339 (1979), utilized the stain disclosed by Switzer, Merril and Shifrin in Anal. Biochem, (1979). Some potential clinical applications were demonstrated as well as that the stain was more sensitive than Coomassie Blue, and less expensive and more rapid than autoradiography.
Oakley, Kirsch and Morris, in "A Simplified Ultrasensitive Silver Stain for Detecting Proteins in Polyacrylamide Gels", Anal. Biochem. 105, 361-363 (1980), which was published less than one year prior to filing the application for this patent in the United States, discloses an adaptation of the silver stain first disclosed by Switzer, Merril, and Shifrin (1979), supra. The disclosed process utilizes (1) soaking the gel in glutaraldehyde for 30 minutes, (2) rinsing and soaking the gel in water for at least 2 hours, (3) adding ammoniacal silver solution (a mixture of NH.sub.4 OH, NAOH, and AgNO.sub.3), (4) transferring the gel to a mixture of citric acid and formaldehyde, and (6) washing in water for at least 1 hour. The stated advantages are simplification of the original procedure, elimination of the cupric-silver nitrate step, and reduction of the amount of silver required. In an attempted replication of this method in connection with the present invention, a significant reduction (about 50%) in sensitivity was observed when the stain method of Oakley, Kirsch and Morris, supra, was compared with that of the improved method of this invention.
No patents are known which disclose silver stains for proteins or which are more relevant than the foregoing monographs.
U.S. Pat. No. 3,873,433 does not disclose silver stains, but does disclose protein staining by the formation of complex organic salts with bivalent elements such as calcium and magnesium.
U.S. Pat. No. 4,167,467 does not disclose silver stains, but does disclose the quantification of lipoprotein free cholesterols using a cholesterol oxidase substrate by enzymatic determination.